Berbamine derivatives

ABSTRACT

The invention provides novel berbamine derivatives, and compositions or pharmaceutical compositions thereof. These berbamine derivatives have shown higher potency in killing cancer/tumor cells comparing to berbamine, and can be used in cancer/tumor treatments.

PRIORITY STATEMENT

The present application is a divisional of U.S. application Ser. No.12/783,535, filed May 19, 2010, which claims priority to U.S.Provisional Patent Application No. 61/179,504, filed on May 19, 2009,which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the field of compounds and pharmaceuticalcompositions thereof, and methods of using the compounds andpharmaceutical compositions thereof for treating cancer and tumor.

BACKGROUND OF THE INVENTION

Berbamine is a natural product derived from the plant, Berberisvulgaris, which has been used extensively in Asia and Europe for thetreatment of various ailments. Berbamine is known to possess anticancerproperties. Despite the widespread use of berbamine in traditional plantmedicines, only a handful of berbamine derivatives have been reported.Therefore, there is a need to synthesize novel berbamine derivatives forpotential therapeutic treatments.

SUMMARY OF THE INVENTION

One aspect of the present disclosure relates to berbamine derivativesand pharmaceutical compositions thereof.

Another aspect of the present disclosure relates to a method of treatingcancer and tumor using berbamine derivatives and pharmaceuticalcompositions thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Viability of DU145 cells treated with BA #1 (diamond), BA #2(square), BA #3 (triangle) and BA #4 (—X—).

FIG. 2. Viability of DU145 cells treated with BA #5 (grey block), BA #6(dark block) and phthaloyl derivative (Phthaloyl, white block).

FIG. 3. Viability of DU145 cells treated with BA #7 (dark block), BA #8(white block), BA #9 (stripe block) and BA #10 (grey block).

FIG. 4. Viability of DU145 cells treated with BA #11 (grey block), BA#12 (dark block) and BA #13 (white block).

FIG. 5. Viability of A2058 cells treated with BA #1 (diamond), BA #2(square), BA #3 (triangle) and BA #4 (—X—).

FIG. 6. Viability of A2058 cells treated with BA #5 (grey block), BA #6(dark block) and phthaloyl derivative (Phthaloyl, white block).

FIG. 7. Viability of A2058 cells treated with BA #7 (dark block), BA #8(white block), BA #9 (stripe block) and BA #10 (grey block).

FIG. 8. Viability of A2058 cells treated with BA #11 (grey block), BA#12 (dark block) and BA #13 (white block).

FIG. 9. Viability of K562/ADR cells treated with BA #1 (circle), BA #3(triangle) and BA #4 (square).

FIG. 10. Viability of human normal dermal fibroblast (NHDF) cellstreated with berbamine (dark block), BA #3 (white block), BA #10 (stripeblock) and BA #12 (grey block).

FIG. 11. Viability of human erythroleukemia HEL (homozygous Jak2 V617Fmutation) cells treated with berbamine (dark block), BA #3 (whiteblock), and BA #10 (stripe block).

FIG. 12. BA #3 reduced cell viability in cancer cells. A) in human acutelymphoblastic leukemia (Molt4) cells (grey block), human acutelymphocytic leukemia (Reh) cells (dark block), and multiple myeloma(U266) cells (white block); B) in melanoma cells: A375 (dark block),G361 (white block), SK-MEL-28 (stripe block) and SK-MEL-5 (grey block).

FIG. 13. BA #3 reduced cell viability in cancer cells.

FIG. 14. BA #3 inhibited Jak2/Stat3 signaling: A) in A2058 melanomacells after 4 hour treatment of different concentrations of BA #3 (0-10μM); B) in A2058 melanoma cells after different time of treatment (0-24hour) of 10 μM BA #3; and C) in the erythroleukemia HEL (HEL Jak2 V617Fmutation) cells after 4 hour treatment of different concentrations of BA#3 (0-10 μM).

FIG. 15. In vitro Src kinase assay of BA #3.

FIG. 16. In vitro Jak2 kinase assay of BA #3.

FIG. 17. In vitro Jak1 kinase assay of BA #3.

FIG. 18. In vitro Trk2 kinase assay of BA #3.

FIG. 19. In vitro Jak2 autophosphorylation kinase assay of BA #3. A)Autophosphorylation of Jak2 at Tyr1007/1008 was required for itscatalytic activity, non-activated recombinant Jak2 protein was mixedwith ATP in the absence or presence of BA #3; B) inhibitory activity ofJak2 autophosphorylation based on the densitometry of FIG. 19(A).

FIG. 20. BA #3 down-regulated Mcl-1 and Bcl-x_(L) proteins.

FIG. 21. BA #3 induced apoptosis in A2058 cells.

FIG. 22. Maximum tolerated dose (MTD) study of BA #3 on mice. Bodyweight of mice treated with 50 mg/Kg of BA #3 (square) or 100 mg/Kg ofBA #3 (triangle) were compared with body weight of mice treated withvehicle only (diamond). A) and B) are two sets of experiments carriedout under the same condition.

FIG. 23. BA #3 inhibited Jak2/Stat3 signaling in cells after treatmentof various concentrations of BA #3 for four hours.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present disclosure related to berbamine derivatives.

Berbamine has a structure as shown below:

In certain embodiments, a berbamine derivative has a structurecomprising Structure A:

including pharmaceutically acceptable variants thereof, wherein:

-   -   R is selected from the group consisting of —C(═O)—(CH₂)_(n)NR′R″        and —(CH₂)_(n)NR′R″;    -   R′ is selected from the group consisting of H, substituted and        unsubstituted alkyl, substituted and unsubstituted aryl, and        substituted and unsubstituted acyl groups;    -   R″ is selected from the group consisting of H, substituted and        unsubstituted alkyl, substituted and unsubstituted aryl, and        substituted and unsubstituted acyl groups; and    -   n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

In certain embodiments, R′ and R″ together form a ring structure. Incertain embodiments, R′ and R″ can be the same or different.

As used herein, unless specified otherwise, the term “alkyl” means abranched or unbranched, saturated or unsaturated, monovalent ormultivalent hydrocarbon group, including saturated alkyl groups, alkenylgroups and alkynyl groups. Examples of alkyl include, but are notlimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, ethenyl,propenyl, butenyl, isobutenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, undecenyl, dodecenyl, ethynyl, propynyl, butynyl,isobutynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl,undecynyl, dodecynyl, methylene, ethylene, propylene, isopropylene,butylene, isobutylene, t-butylene, pentylene, hexylene, heptylene,octylene, nonylene, decylene, undecylene and dodecylene. In certainembodiments, the hydrocarbon group contains 1 to 30 carbons. In certainembodiments, the hydrocarbon group contains 1 to 20 carbons. In certainembodiments, the hydrocarbon group contains 1 to 12 carbons.

As used herein, unless specified otherwise, the term “aryl” means achemical structure comprising one or more aromatic rings. In certainembodiments, the ring atoms are all carbon. In certain embodiments, oneor more ring atoms are non-carbon, e.g. oxygen, nitrogen, or sulfur(“heteroaryl”). Examples of aryl include, without limitation, phenyl,benzyl, naphthalenyl, anthracenyl, pyridyl, quinoyl, isoquinoyl,pyrazinyl, quinoxalinyl, acridinyl, pyrimidinyl, quinazolinyl,pyridazinyl, cinnolinyl, imidazolyl, benzimidazolyl, purinyl, indolyl,furanyl, benzofuranyl, isobenzofuranyl, pyrrolyl, indolyl, isoindolyl,thiophenyl, benzothiophenyl, pyrazolyl, indazolyl, oxazolyl,benzoxazolyl, isoxazolyl, benzisoxazolyl, thiaxolyl, quanidino andbenzothiazolyl.

As used herein, the term “acyl” means R₁—C(═O)—, wherein R₁ is asubstituted or unsubstituted alkyl, or a substituted or unsubstitutedaryl group.

As used herein, the term “pharmaceutically acceptable variants” of acompound means variants that retain the biological effectiveness of thecompound itself. Examples of pharmaceutically acceptable variants of acompound include, without limitation, pharmaceutically acceptable saltsof the compound which are well known to those of skill in the art (e.g.hydrochloride, methanesulfonate, mesylate, maleate, decanoate,enanthate, succinate, lactate, sulfate, and quaternary ammonium salts).Another example of pharmaceutically acceptable variants of a compound isa complex of the compound with nanoparticles wherein the complex isformed via covalent and/or non-covalent interactions among the compoundand the nanoparticles.

In certain embodiments, a berbamine derivative has a structurecomprising Structure A, wherein:

n=3;

R is —(CH₂)_(n)NR′R; and

NR′R″ is selected from the group consisting of NH₂,

In certain embodiments, a berbamine derivative has a structurecomprising Structure A, wherein:

n=2;

R is —C(═O)—(CH₂)_(n)NR′R″; and

NR′R″ is selected from the group consisting of NH—C(═O)—O—C(CH₃)₃, andNH₂.

Examples of berbamine derivative include, without limitation, BA #1, BA#2, BA #3, BA #4, BA #5, BA #6, BA #7, BA #8, BA #9, BA #10, BA #11, BA#12, and BA #13 as shown below:

As used herein, the term “phthaloyl derivative” means a compound havingthe following structure:

As used herein, unless specified otherwise, the term “berbamine amidederivative” means a berbamine derivative having a structure comprisingStructure A as described supra, wherein —OR comprises at least one amidefunctional group. Examples of berbamine amide derivatives include,without limitation, BA #1, BA #3, and BA #5-BA #13.

As used herein, unless specified otherwise, the term “berbamine aminederivative” means a berbamine derivative having a structure comprisingStructure A as described supra, wherein —OR comprises at least one aminefunctional group, and the amine group can be primary amine, secondaryamine or tertiary amine group. Examples of berbamine amine derivativesinclude, without limitation, BA #2 and BA #4

As used herein, unless specified otherwise, the term “berbamine esterderivative” means a berbamine derivative having a structure comprisingStructure A as described supra, wherein —OR comprises at least one esterfunctional group. Examples of berbamine ester derivatives include,without limitation, BA #1 and BA #2.

Another aspect of the present disclosure relates to a pharmaceuticalcomposition comprising at least one berbamine derivative and apharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical composition may also comprise other known cancer drugs.

The term “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a berbamine derivativefrom one location, body fluid, tissue, organ (interior or exterior), orportion of the body, to another location, body fluid, tissue, organ, orportion of the body.

Each carrier is “pharmaceutically acceptable” in the sense of beingcompatible with the other ingredients, e.g., a berbamine derivative, ofthe formulation and suitable for use in contact with the tissue or organof a biological system without excessive toxicity, irritation, allergicresponse, immunogenicity, or other problems or complications,commensurate with a reasonable benefit/risk ratio.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) alcohol, such as ethyl alcohol and propane alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations such as acetone.

The pharmaceutical compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like.

In one embodiment, the pharmaceutically acceptable carrier is an aqueouscarrier, e.g. buffered saline and the like. In certain embodiments, thepharmaceutically acceptable carrier is a polar solvent, e.g. water,acetone and alcohol.

The concentration of berbamine derivative in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the biological system's needs. Forexample, the concentration can be from about 50 mg/kg to about 100mg/kg, from about 0001% to about 100% wt, from about 0.001% to about 50%wt, from about 0.01% to about 30% wt, or from about 0.1% to about 10%wt. The compositions of the invention can be administered fortherapeutic use. Such administration can be topical, mucosal, e.g.,oral, nasal, vaginal, rectal, parenteral, transdermal, subcutaneous,intramuscular, intravenous, via inhalation, ophthalmic and otherconvenient routes. The pharmaceutical compositions can be administeredin a variety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and lozenges.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 10⁻¹⁰ g to about 100 g, about 10⁻¹⁰ g toabout 10⁻³ g, about 10⁻⁹ g to about 10⁻⁶ g, about 10⁻⁶ g to about 100 g,about 0.001 g to about 100 g, about 0.01 g to about 10 g, or about 0.01g to about 1 g per subject per day. Dosages from about 0.01 mg, up toabout 50 g, per subject per day may be used.

Another aspect of the present disclosure relates to a method of treatingcancer or tumor using a berbamine derivative, or a composition thereof.

Examples of cancers and tumors include, without limitation, benigntumors, solid tumors, breast cancer, SCCHN, renal cell carcinoma,colon-rectal cancer, oral cancer, lung or other respiratory systemcancers, melanoma, skin cancers, uterine cancer, pancreatic cancer,pancreatic adenocarcinoma, liver cancer, prostate cancer, prostatecarcinoma, cervical cancer, testicular cancer, genital cancer, bladdercancer, kidney cancer, urinary organs cancers, ovarian cancer, ovariancarcinoma, leukemia (e.g. HTLV-I-dependent leukemia, erythroleukemia,acute lymphoblastic leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, megakaryotic leukemia,and large granular lymphocyte leukemia), multiple myeloma, lymphomas(e.g. EBV-related/Burkitt's lymphomas, mycosis fungoides, HSVsaimiri-dependent (T-cell) lymphomas, and Hodgkin's disease), bloodtumors, and blood and lymphaptic tissues cancers.

Examples of berbamine derivatives have shown stronger activity thanberbamine in killing cancer and tumor cells, e.g. prostate cancer (e.g.DU145) cells, melanoma (A2058, A375, G361, SK-MEL-28 and SK-MEL-5)cells, human acute lymphoblastic leukemia (Molt4) cells, human acutelymphocytic leukemia (Reh) cells, imatinib-resistant leukemia (e.g.K562/ADR) cells, multiple myeloma (e.g. U266) cells, humanerythroleukemia HEL (homozygous JAK2 V617F mutation) cells, human normaldermal fibroblast (NHDF) cells, ovarian cancer (OVCAR5, SKOV3) cells andpancreatic cancer (PANG-1) cells.

IC₅₀ values of examples of berbamine derivatives range from about 1 μMto about 16 μM, while IC₅₀ values of berbamine is >20 μM. For example,berbamine derivatives (e.g. BA #3) show significant (at least 10-fold)improvement in activity over berbamine in cancer cell lines tested (e.g.K562/ADR). Berbamine derivatives (e.g. BA #3) also show inhibition ofJak2/Stat3 signaling in cancer cell lines (e.g. A2058, erythroleukemiaHEL cells), and show down-regulation of Mcl-1 and Bcl-x_(L) proteins.Berbamine derivatives (e.g. BA #3) show induction of apoptosis in cancercell lines (e.g. A2058) and subjects (e.g. mice) treated with berbaminederivatives (e.g. BA #3) do not show significant weight differencecomparing to subjects not treated with any berbamine derivatives.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted in any way as limiting the scopeof the invention. All specific compositions, materials, and methodsdescribed below, in whole or in part, fall within the scope of theinvention. These specific compositions, materials, and methods are notintended to limit the invention, but merely to illustrate specificembodiments falling within the scope of the invention. One skilled inthe art may develop equivalent compositions, materials, and methodswithout the exercise of inventive capacity and without departing fromthe scope of the invention. It will be understood that many variationscan be made in the procedures herein described while still remainingwithin the bounds of the invention. It is the intention of the inventorsthat such variations are included within the scope of the invention.

Example 1 Preparation of Berbamine Derivatives

Synthesis of BA #3:

BA #3 can be prepared using a synthesis route as shown in Scheme 1. Inone embodiment, berbamine (1 eq) in DMF (0.2M) was added to a suspensionof degreased NaH (3 eq) in DMF (0.5M) at 0° C. The obtained suspensionwas slowly warmed to room temperature, stirred for 2 hours, and cooledto 0° C. Then bromophthalimide (1.5 eq) was added to the suspension, andthe reaction mixture was warmed to room temperature and stirredovernight. Saturated aqueous NH₄Cl solution was added to the reactionmixture, and then the reaction mixture was poured into water andextracted with ethyl acetate (3×). The organic layer was washed withwater (2×), dried over MgSO₄, filtered and concentrated. The crudemixture was purified via SiO₂ using 4% NH₃/MeOH in dichloromethane(DCM), and the yield was ˜50%.

Synthesis of BA #4:

BA #4 can be prepared using a synthesis route as shown in Scheme 2. Inone embodiment, hydrazine-hydrate (50 eq) was added to a solution ofberbamine-phthalimide (1 eq) in EtOH (0.1M). The obtained reactionmixture was refluxed overnight with stirring. The reaction mixture wasthen cooled to room temperature and poured into water/DCM. The aqueouslayer was extracted 2× with additional DCM, and the combined organiclayer was dried over MgSO₄, filtered and concentrated. No furtherpurification was needed, and the yield was ˜85%.

Synthesis of Berbamine Amide Derivatives (e.g. BA #5-#13):

Berbamine amide derivatives can be prepared by reacting a berbamineamine derivative with a desired acid or a desired acid chloride(exemplary synthesis route as shown in Scheme 3). The berbamine aminederivative can be prepared as described supra (exemplary synthesis routeas shown in Scheme 2).

In one embodiment, an aryl acid chloride (2 eq) was slowly added to a 0°C. solution of a desired berbamine amine derivative (1 eq) and distilledNEt₃ (4 eq) in THF (0.1M). The reaction mixture was warmed to roomtemperature and stirred overnight. Then, the reaction solution waspoured into water and extracted with EtOAc (2×). The combined organiclayer was washed with water, dried over MgSO₄, filtered, concentratedand further purified via SiO₂ using 3% NH₃/MeOH in DCM, and the yieldwas ˜50-75%.

In one embodiment, a desired berbamine amine derivative (1 eq), adesired aryl acid (1.5 eq), EDCI-HCl (1.5 eq) and DMAP (0.1 eq) werecombined in DMF (0.1M) and stirred at room temperature overnight. Thereaction mixture was poured into water and extracted with EtOAc (3×).The combined organic layer was washed with water (2×), dried over MgSO₄,filtered, concentrated, and further purified via SiO₂ using 3% NH₃/MeOHin DCM. The yield was 60-80%.

Synthesis of Berbamine Ester Derivatives (e.g. BA #1 and BA #2)

Berbamine ester derivatives can be prepared by reacting berbamine or aberbamine derivative having a hydroxyl group with a desired acid or acidchloride.

For example, BA #1 was prepared by reacting berbamine with a desiredacid in the presence of DCC (Scheme 4). BA #2 was prepared by hydrolysisof BA #1 (Scheme 4).

Example 2 Berbamine Derivatives Showed Lower IC₅₀ in DU145 ProstateCancer Cell Line, A2058 Human Melanoma Cell Line Comparing to Berbamine

IC₅₀ of values of berbamine derivatives were determined in cancer celllines DU145 and A2058 respectively. DU145 and A2058 cells (5,000cells/well) were treated with berbamine derivatives in a dose-dependentmanner (0.1 to 20 μM in 1% DMSO for 48 h. Then, MTS assay for cellviability were carried out to determine the IC₅₀ values. Four sampleswere tested for each berbamine derivatives at each concentration in eachcell line, and the averaged data were shown in Table 1 and FIGS. 1-8.

TABLE 1 IC₅₀ of berbamine derivatives in cancer cell lines DU 145 andA2058 Test Cell line DU145 A2058 Berbamine IC₅₀ (μM) >20 >20 Phthaloylderivative IC₅₀ (μM) >20 >20 BA #1 IC₅₀ (μM) 9.4 7.4 BA #2 IC₅₀ (μM)15.2 14.6 BA #3 IC₅₀ (μM) 3.4 2.9 BA #4 IC₅₀ (μM) 15.5 14.3 BA #5 IC₅₀(μM) 7.9 3.9 BA #6 IC₅₀ (μM) 3.5 3.4 BA #7 IC₅₀ (μM) 6.9 3.5 BA #8 IC₅₀(μM) 6.6 7.1 BA #9 IC₅₀ (μM) 7.7 8.1 BA #10 IC₅₀ (μM) 3.7 3.4 BA #11IC₅₀ (μM) 12.3 8.3 BA #12 IC₅₀ (μM) 5.6 3.3 BA #13 IC₅₀ (μM) 13.6 8.2

Example 3 Berbamine Derivatives Showed Lower IC₅₀ in Imatinib-ResistantLeukemia (K562/ADR) Cells Comparing to Berbamine

Imatinib-resistant leukemia (K562/ADR) cells (5,000 cells/well) weretreated with different concentrations (0.6-40 μM in DMSO) of BA #1, BA#3 and BA #4 respectively for 48 hours and cell viability was measured.The results (Table 2, FIG. 9) indicated that berbamine derivativesdisplayed higher potency to kill K562/ADR cells comparing to berbamine.

TABLE 2 IC₅₀ of berbamine derivatives in K562/ADR Berbamine BA #1 BA #3BA #4 IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) K562/ADR 8.9 6.4 1.1 3.0

Example 4 Berbamine Derivatives Showed Lower IC₅₀ Human Normal DermalFibroblast (NHDF) Cell Line and Human Erythroleukemia HEL (HomozygousJAK2 V617F Mutation) Cells Comparing to Berbamine

Human normal dermal fibroblast (NHDF) (5,000) cells were treated withdifferent concentration (0.1 μM to 100 μM in 1% DMSO of berbamine, BA#3, BA #10 and BA #12 respectively for 48 hours and cell viability wasmeasured. The results (Table 3, FIG. 10) indicated that berbaminederivatives displayed higher potency to kill NHDF cells comparing toberbamine.

HEL Jak2 V617 mutation cells (10,000 cells/well) were treated withdifferent concentration (0.1 μM to 20 μM in 1% DMSO of berbamine, BA #3,and BA #10 respectively for 48 h. Then, MTS assay for cell viabilitywere carried out to determine the IC₅₀ values. Four samples were testedfor each test compound at each concentration, and the averaged data wereshown in Table 3 and FIGS. 10-11.

TABLE 3 IC₅₀ of berbamine derivatives in NHDF cells and HEL cells.Berbamine BA #3 BA #10 BA #12 IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM)NHDF 24 7.5 9.4 9.2 HEL Jak2 V617 19.6 3.2 2.8 N/A mutation

Example 5 BA #3 Reduced Cell Viability in Human Acute LymphoblasticLeukemia (Molt4) Cells, Human Acute Lymphocytic Leukemia (Reh) Cells,Multiple Myeloma (U266) Cells, Melanoma (A375, G361, SK-MEL-28 andSK-MEL-5) Cells, Ovarian Cancer (OVCAR5, SKOV3) Cells and PancreaticCancer (PANC-1) Cells

Each type of cells (5,000 or 10,000 cells/well) were treated withdifferent concentration (0.1 μM to 20 μM in 1% DMSO of berbamine and BA#3 respectively for 48 h. Then, MTS assay for cell viability werecarried out to determine the IC₅₀ values. Four samples were tested foreach test compound at each concentration for each type of cells, and theaveraged data were shown in Table 4 and FIGS. 12-13.

TABLE 4 IC₅₀ of berbamine and BA #3 in Molt4 cells, Reh cells, U266cells, A375 cells, G361 cells, SK-MEL-28 cells and SK-MEL-5 cells SK-Test MEL- SK- cells Molt4 Reh U266 A375 G361 28 MEL-5 OVCAR5 SKOV3PANC-1 IC₅₀ 3.9 2.9 6.7 3.7 3.1 3.0 3.3 2.8 3.9 6.4 (μM)

Example 6 BA #3 Inhibited Jak2/Stat3 Signaling in A2058 Melanoma Cellsand Erythroleukemia HEL Cells

BA #3 inhibited Jak2/Stat3 signaling in A2058 melanoma cells (FIGS.14(A) and (B)) and erythroleukemia HEL cells (FIG. 14(C)). To determinewhether BA #3 block Jak2/Stat3 or Src/Stat3 signaling pathway, A2058melanoma (A and B) and erythroleukemia HEL cells were treated with BA #3in a dose- or time-dependent manner. Western blots were performed withspecific antibodies to p-Jak2, p-Src, p-Stat3, p-AKT, Jak2, Src, Stat3and AKT. As shown in Figures, BA #3 inhibited phosphorylations of Jak,Src, Stat3 and AKT, indicating BA #3 blocks Jak2/Stat3 or Src/Stat3.

Example 7 BA #3 Reduced Jak2 Autophosphorylation Activities atTyr1007/1008 Sites in Vitro

To determine whether BA #3 inhibits activities of Src kinase, Jak2kinase, Jak1 kinase and Tyk2 kinase in vitro, activated recombinantproteins were mixed with peptide substrates and ATP in the absence orthe presence of BA #3. BA #3 did not inhibit these kinase activities invitro with substrates (FIGS. 15, 16, 17 and 18). However, BA #3 reducedJak2 autophosphorylation kinase activity at Tyr1007/1008 sites in vitroin the reaction of mixture of non-activated recombinant Jak2 protein andATP in the absence of substrates (FIG. 19).

BA #3 did not inhibit activities of Src kinase (FIG. 15), Jak2 kinase(FIG. 16), Jak1 kinase (FIG. 17) and Tyk2 kinase (FIG. 18) withsubstrates in vitro.

IC₅₀ of BA #3 for inhibition of autophosphorylation kinase activity invitro was 0.69 μM (FIG. 19).

Example 8 BA #3 Down-Regulated Mcl-1 and Bcl-xL Proteins

To determine whether BA #3 down-regulates Stat3 downstream anti-apototicproteins Mcl-1 and Bcl-xL, cells were treated with BA #3 in adose-dependent manner for 24 h. Western blots were performed withspecific antibodies to Mcl-1 and Bcl-xL. The results showed that BA #3down-regulated Mcl-1 and Bcl-xL at 5 μM concentration (FIG. 20).

Example 9 BA #3 Induced Apoptosis in A2058 Cells

A2058 cells were treated with BA #3 in a dose dependent manner (0-10 μM)for 48 hours. Apoptosis assay was carried out using Annexin V-FITC, andthe results showed that BA #3 induced apoptosis in A2058 cells (FIG.21).

Example 10 Maximum Tolerated Dose (MTD) Study of BA #3 in Mice

Three test groups of mice (four mice per group) were orallyadministrated with vehicle only, 50 mg/kg BA #3 and 100 mg/kg BA #3respectively every day for 5 days. Mice were weighed every day. Two setsof experiments were carried out (FIGS. 22(A) and (B)). The resultsshowed no significant weight differences between the mice treated withvehicle only and the mice treated with BA #3.

The invention claimed is:
 1. A compound comprising the followingstructure of Structure A

or a pharmaceutically acceptable variant thereof, wherein: R is selectedfrom the group consisting of —C(═O)—(CH₂)_(n)NR′R″ and —(CH₂)_(n)NR′R″;R′ is selected from the group consisting of H, substituted andunsubstituted alkyl, and substituted and unsubstituted acyl groups; R″is selected from the group consisting of H, substituted andunsubstituted alkyl, and substituted and unsubstituted acyl groups; n is2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30; and when n is 2, and R is—(CH₂)_(n)NR′R″, at least one of R′ and R″ is not H.
 2. A pharmaceuticalcomposition comprising a compound according to claim 1 and apharmaceutically acceptable carrier.
 3. A method of treating cancer andtumor comprising administering a therapeutically effective amount of apharmaceutical composition comprising a compound according to claim 1.4. The method according to claim 3, wherein the cancer and tumor isselected from the group consisting of benign tumor, solid tumor, breastcancer, colon-rectal cancer, oral cancer, lung cancer, respiratorysystem cancers, melanoma, skin cancers, uterine cancer, pancreaticcancer, liver cancer, prostate cancer, cervical cancer, testicularcancer, genital cancer, bladder cancer, kidney cancer, urinary organscancers, ovarian cancer, leukemia, acute lymphoblastic leukemia, acutelymphocytic leukemia, erythroleukemia, multiple myeloma, blood cancer,and lymphatic tissues cancer.
 5. The method according to claim 4,wherein the compound has a structure selected from the group consistingof BA #1, BA #2, BA #3, BA #4, BA #5, BA #6, BA #7, BA #8, BA #9, BA#10, BA #11, BA #12, and BA #13: